Bacillus anthracis is a spore-forming bacterium that causes the disease anthrax with lethality near 100% when infections are initiated by the inhalation of spores. The traditional model of how inhalational infections initiate is the Trojan horse model. In this model spores that are inhaled into the alveoli are phagocytosed by resident phagocytes and carried across the epithelial barrier intracellularly to the draining mediastinal lymph nodes (MLN) where they germinate, escape from the phagocyte, grow within the lymph node, and disseminate to the blood system. Mounting evidence from our lab and others suggest that aspects of this model should be revisited. Tracking the growth and spread of bioluminescent bacteria by imaging (BLI) within a mouse model of inhalational infection demonstrated that the earliest bacterial growth takes place not in the MLN, but in the nasal associated lymphoid tissues (NALT). This occurs at a time when only ungermianted spores can be isolated from the MLN. Furthermore, other groups have demonstrated both that phagocytes can destroy B. anthracis and that spores can transcytose through intact airway epithelia, suggesting that B. anthracis transit through phagocytes may be costly to the bacteria and that epithelial invasion may be an alternative. We hypothesize that, though bacteria can be isolated from both the MLN and NALT, the earlier growth of bacteria in the NALT indicates that these bacteria are the population that ultimately cause the lethal disseminated disease. We propose to determine whether the bacteria in the MLN or NALT are the founding population for disseminated lethal anthrax. Such a determination is important since if the bacteria in the MLN are the founders, we will provide the first concrete evidence that fatal inhalational anthrax originates via a pulmonary portal of infection. On the other hand, if the NALT is the portal of entry that leads to fatal disease, as we predict, a major shift in the field of anthrax research must occur to reduce the focus on alveolar phagocyte transport, and instead refocus on the NALT. If this is the case, the reasons why the alveolar interactions tend to control B. anthracis while NALT interactions do not will be of profound importance for designing prophylactic treatments for inhalational anthrax. Identifying the founding population will be achieved through the use of a technique known as "clonal analysis". The clonal analysis will consist of constructing a library of equally virulent bioluminescent B. anthracis clones that each have a unique identifying tag and then using this library in a mouse model of inhalational anthrax. As more fully described in Experimental Approach, the unique set of clones found within the MLN and NALT will be compared to the set of clones isolated from deeper organs infected by the disseminated bacteria, such as the kidney and spleen. If the set of clones found within the kidney and spleen is identical to the MLN, this would indicate that the population in the deep organs originated in the MLN. Likewise, if the set of clones in the kidney and spleen are identical to the NALT, this implies that the NALT bacteria were the founding bacteria of the disseminated disease. PUBLIC HEALTH RELEVANCE: According to the long-standing Trojan horse model of infection the spore-forming bacterium Bacillus anthracis causes highly lethal inhalational anthrax when airborne spores enter the mammalian airways and are carried by resident lung defense cells into deeper tissues inside the body. Based on our recent findings we hypothesize instead that B. anthracis spores initiate inhalational infection in the nasal passages and aim to determine whether this is true by genetically tagging bacteria to determine whether the bacteria found in deep tissues came from the nasal passages or lungs. If the nasal passages prove to be the portal of entry a major shift in the field of anthrax research should occur to reduce the focus on preventing lung invasion and instead realign to concentrate on blocking spores from invading the nasal passages.